Comparison of Insect Gut Cellulase and Xylanase Activity Across Different Insect Species with Distinct Food Sources

Insect guts represent unique natural biocatalyst systems for biocatalyst discovery and biomass deconstruction mechanism studies. In order to guide the further research for enzyme discovery and biodiversity analysis, we carried out comprehensive xylanase and cellulase activity assays for the gut contents of three insect species representing different orders and food sources. The three insect species are grasshopper (Acrididae sp.), woodborer (Cerambycidae spp.), and silkworm (Bombyx mori) to represent the wood-consuming, grass-consuming, and leaf-consuming insects from Orthoptera, Coleoptera, and Lepidoptera orders, respectively. Generally speaking, the enzyme activity assays have shown that the cellulase and xylanase activities for grasshopper and woodborer guts are significantly higher than those of silkworm under various conditions. In addition, both pH and temperature have a significant impact on the enzyme activities in the gut contents. For the grasshopper gut, the means of xylanase and cellulase activities at pH 7 were 3,397 and 404 μM mg^?1 min^?1, which are significantly higher than the activities at pH 4 and 10 (P?<?0.05). However, woodborer guts have shown the highest cellulase activity at pH 10. The results suggested that systems similar to woodborer guts could be good resources for discovering alkaline-tolerant enzymes. Moreover, the enzyme activities in response to different substrate concentrations were also analyzed, which indicated that grasshopper gut had particularly high cellulase activity. The enzyme activities in response to the reaction time were also examined, and we found that the enzyme activities (micromolar per milligram per minute) of different insect gut juices in response to the increase of incubation time fit well to the power function equation (E _c = K ? t ^b ) with high coefficients (r ²?>?0.99). The newly developed model serves well to compare the characteristics of the enzyme mixtures among different insect species, which can be applied to other studies of natural biocatalyst systems for the future. Overall, the data indicated that grasshopper and woodborer guts are valuable resources for discovering the novel biocatalysts for various biorefinery applications.     

Stability analysis of liver cancer-related microRNAs

MicroRNAs (miRNAs) are non-coding, single-stranded RNAs of approximately 22 nt and constitute a novel class of gene regulators that are found in both plants and animals. Several studies have demonstrated that serum miRNAs could serve as potential biomarkers for the detection of various cancers and other diseases. A few documents regarding the stability of liver cancer-related miRNAs in serum are available. A systemic analysis of the stability of miRNA in serum is quite necessary. The purpose of this study was to evaluate the stability of miRNAs from three different sources, cultured liver cancer Huh-7 cell line, clinical liver cancer, and serum under different experimental conditions, including different temperature, time duration, pH values, RNase A digestion, DNase I digestion, and various freeze-thaw cycles. The qRT-PCR analysis demonstrated that liver cancer-related miRNAs were detectable under each of test conditions, indicating that miRNAs were extremely stable and resistant to destruction and degradation under harsh environmental conditions. However, ribosomal RNA was fragile and easily degraded by demonstrating sharp decrease of relative expression under the non-physiological test conditions. We also established a robust procedure for serum RNA extraction, which is greatly important not only for the miRNA profiling studies but also for the disease prognosis based on abnormal miRNA expression.     

Evolution of vertebrate central nervous system is accompanied by novel expression changes of duplicate genes

The evolution of the central nervous system (CNS) is one of the most striking changes during the transition from invertebrates to vertebrates. As a major source of genetic novelties, gene duplication might play an important role in the functional innovation of vertebrate CNS. In this study, we focused on a group of CNS-biased genes that duplicated during early vertebrate evolution. We investigated the tempo-spatial expression patterns of 33 duplicate gene families and their orthologs during the embryonic development of the vertebrate Xenopus laevis and the cephalochordate Brachiostoma belcheri. Almost all the identified duplicate genes are differentially expressed in the CNS in Xenopus embryos, and more than 50% and 30% duplicate genes are expressed in the telencephalon and mid-hindbrain boundary, respectively, which are mostly considered as two innovations in the vertebrate CNS. Interestingly, more than 50% of the amphioxus orthologs do not show apparent expression in the CNS in amphioxus embryos as detected by in situ hybridization, indicating that some of the vertebrate CNS-biased duplicate genes might arise from non-CNS genes in invertebrates. Our data accentuate the functional contribution of gene duplication in the CNS evolution of vertebrate and uncover an invertebrate non-CNS history for some vertebrate CNS-biased duplicate genes.     

Allelopathy of diterpenoids on three species of soil ciliates

Allelopathy of diterpenoids extracted from plants of the genus Robdosia on three common species of soil ciliates, Colpoda inflata, Colpoda cucullus and Euplotes muscicola, was studied by acute toxicity test, sub-lethal effect test and morphological observation. Acute toxicity test showed that there was remarkable toxicity of the diterpenoids on the individuals of the three soil ciliate species, and there was close correlation between toxicity and concentration of the diterpenoids. 12 h-LC₅₀ values of the diterpenoids on the individuals of C. inflata, C. cucullus and E. muscicola were 161.40 mg L^?1, 94.80 mg L^?1 and 83.70 mg L^?1 respectively, and 24 h-LC₅₀ values were 114.90 mg L^?1, 92.30 mg L^?1and 65.80 mg L^?1 separately. Sub-lethal effect test of soil ciliates suggested that there existed significant inhibition of the diterpenoids on population growth of the three ciliates with dose-dependant relationships, population density and growth rate of the test group was obviously lower than that of the control group. Morphological observation indicated that diterpenoids affected the body shapes of the three ciliates and made them shorter and thicker, and the higher the concentration of diterpenoids, the greater the affection. The results are of great significance for understanding the functions of ciliates and their relationships to other organisms, and for the application of allelopathy in biological pest control in the soil ecosystems.     

CCN-2 is up-regulated by and mediates effects of matrix bound advanced glycated end-products in human renal mesangial cells

CCN-2, also known as connective tissue growth factor (CCN-2/CTGF) is a cysteine rich, extracellular matrix protein that acts as a pro-fibrotic cytokine in tissues in many diseases, including in diabetic nephropathy. We have published that soluble advanced glycation end products (AGEs), that are present in increased amounts in diabetes, induce CCN-2. However in vivo AGEs are known to be heavily tissue bound and whether matrix bound AGEs regulate CCN-2 has not been investigated. In this study we determined in human renal mesangial cells if CCN-2 is induced by matrix associated AGEs and if CCN-2 may then secondarily mediate effects of matrix AGEs on extracellular matrix expansion. Data generated show that CCN-2 mRNA and protein expression are induced by matrix bound AGEs, and in contrast, this was not the case for TGF-β1 mRNA regulation. Using CCN-2 adenoviral anti-sense it was found that CCN-2 mediated the up-regulation of fibronectin and the tissue inhibitor of matrix metalloproteinase, TIMP-1, that was caused by matrix bound AGEs. In conclusion, CCN-2 is induced by non-enzymatically glycated matrix and it mediates downstream fibronectin and TIMP-1 increases, thus through this mechanism potentially contributing to ECM accumulation in the renal glomerulus in diabetes.